Animals with reduced body fat and increased bone density

ABSTRACT

Methods for identifying animals as having reduced body fat and increased bone density are provided herein. Also provided herein are methods for generating animals having reduced body fat and increased bone density. The methods provided herein are based on the effect of TLR4, MD-2, and CD14 activity on body fat and bone density.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. application Ser. No. 10/543,874, filed on Jul. 12, 2006, which is a National Stage application under 35 U.S.C. §371 and claims benefit under 35 U.S.C. §119(a) of International Application No. PCT/US2005/006970 having an International Filing Date of Mar. 3, 2005, which claims the benefit of priority of U.S. Provisional Application Ser. No. 60/550,446 having a filing date of Mar. 5, 2004.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant nos. HL 46810 and AI053733, awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

This invention relates to materials and methods for generating and/or identifying animals with altered TLR4, MD-2, or CD14 activity.

BACKGROUND

The Toll family of proteins is remarkably conserved across the taxonomic kingdoms. This family includes the invertebrate Toll proteins, the vertebrate Toll-like receptors, and the plant resistance genes (Hoffmann and Reichhart (2002) Nat. Immunol. 3:121-126; Akira et al. (2001) Nat. Immunol. 2:675-680; and Hulbert et al. (2001) Annu. Rev. Phytopathol. 39:285-312). Many of these proteins have homologous domains, and play roles in signaling pathways that trigger inflammatory and immunological responses. At least ten Toll-like receptor proteins have been identified. These type I transmembrane proteins are characterized by an extracellular domain with leucine-rich repeats and a cytoplasmic domain with homology to the type I IL-1 receptor. Most Toll-like receptor proteins are expressed on cells of the immune system. The function of these proteins, however, extends beyond host defense.

SUMMARY

The invention is based on the discovery that animals with altered (e.g., reduced) Toll-like receptor 4 (TLR4), MD-2, and/or CD14 activity have reduced body fat and increased bone density. Altered TLR4, MD-2, and/or CD14 activity may result from amino acid sequence variants in the TLR4, MD-2, and/or CD14 polypeptides, which typically result from nucleotide sequence variants in the nucleic acids that encode these polypeptides.

The invention provides methods for identifying animals having reduced body fat and increased bone density. In the methods provided herein, animals are identified based on (a) altered levels of TLR4, MD-2, and/or CD14 activity, or (b) the presence of variant TLR4, MD-2, and/or CD14 polypeptides or nucleic acids. Also provided herein are methods for generating animals having reduced body fat and increased bone density. Such animals can be generated by treatment with one or more agents that result in altered TLR4, MD-2, and/or CD14 activity, or by genetic engineering to generate animals containing TLR4, MD-2, or CD14 sequence variants, for example.

In one aspect, the invention features a method for identifying an animal as having reduced body fat. The method can include (a) determining the level of TLR4, MD-2, or CD14 activity in a biological sample from the animal, and (b) identifying the animal as having reduced body fat if the level of TLR4, MD-2, or CD14 activity is reduced as compared to a control level of TLR4, MD-2, or CD14 activity. The control level of TLR4, MD-2, or CD14 activity can be the level of TLR4, MD-2, or CD14 activity in a corresponding control animal, a standard level of TLR4, MD-2, or CD14 activity, or the average level of TLR4, MD-2, or CD14 activity in a control population of animals. The animal can be bovine, ovine, porcine, or fowl. The level of TLR4, MD-2, or CD14 activity in the biological sample can be determined by measuring the expression of a nucleic acid (e.g., a nucleic acid encoding a cytokine such as an interleukin or TNF-I, or a chemokine such as IP10). The level of TLR4, MD-2, or CD14 activity can be determined by measuring the level of a TLR4, MD-2, or CD14 polypeptide in, for example, serum or tissue. The level of TLR4, MD-2, or CD14 activity can be determined by measuring the level of a small molecule (e.g., prostaglandin E2, leukotriene B(4), or nitric oxide) in the biological sample.

In another aspect, the invention features a method for identifying an animal as having reduced body fat, wherein the method includes determining whether a TLR4, MD-2, or CD14 nucleic acid obtained from the animal contains a variant as compared to a TLR4, MD-2, or CD14 nucleic acid from a corresponding control animal, and identifying the animal as having reduced body fat if the nucleic acid contains the variant. The invention also features a method for identifying an animal as having reduced body fat, wherein the method includes determining whether a TLR4, MD-2, or CD14 polypeptide obtained from the animal contains a variant as compared to a TLR4, MD-2, or CD14 polypeptide from a corresponding control animal, and identifying the animal as having reduced body fat if the polypeptide contains the variant.

In another aspect, the invention features a method for identifying an animal as having increased bone density compared to a corresponding control animal. The method can include (a) determining the level of TLR4, MD-2, or CD14 activity in a biological sample from the animal; and (b) identifying the animal as having increased bone density if the level of TLR4, MD-2, or CD14 activity is reduced as compared to the level of TLR4, MD-2, or CD14 activity in a corresponding control animal.

In still another aspect, the invention features a method for generating an animal having reduced body fat as compared to a corresponding control animal. The method can include (a) identifying a first animal as having a variant TLR4, MD-2, or CD14 nucleic acid or a variant TLR4, MD-2, or CD14 polypeptide, and (b) breeding the first animal with a second animal identified as having a variant TLR4, MD-2, or CD14 nucleic acid or polypeptide, wherein offspring of the breeding exhibit reduced TLR4, MD-2, or CD14 activity as compared to the control animal. The second animal can be identified as having the same variant TLR4, MD-2, or CD14 nucleic acid or polypeptide as the first animal. Alternatively, the second animal can be identified as having a variant TLR4, MD-2, or CD14 nucleic acid or polypeptide that differs from the variant TLR4, MD-2, or CD14 nucleic acid or polypeptide of the first animal. The offspring can have increased bone density as compared to the control animal. The animal can be bovine, ovine, porcine, or fowl.

In another aspect, the invention features a method for generating an animal having reduced body fat as compared to a corresponding control animal. The method can include (a) identifying a first animal as having a reduced level of TLR4, MD-2, or CD14 activity as compared to the corresponding control animal; and (b) breeding the first animal with a second animal identified as having a reduced level of TLR4, MD-2, or CD14 activity as compared to the corresponding control animal, wherein offspring of the breeding exhibit reduced a reduced level of TLR4, MD-2, or CD14 activity as compared to the corresponding control animal. The offspring can have increased bone density as compared to the control animal. The animal can be bovine, ovine, porcine, or fowl. The level of TLR4, MD-2, or CD14 activity in the first or second animal can be determined by measuring the expression of a nucleic acid (e.g., a nucleic acid encoding a cytokine such as interleukin or TNF-I, or a chemokine such as IP 10). The level of TLR4, MD-2, or CD14 activity in the first of second animal can be determined by measuring the level of a TLR4, MD-2, or CD14 polypeptide (e.g., in serum or tissue). The level of TLR4, MD-2, or CD14 activity can be determined by measuring the level of a small molecule (e.g., prostaglandin E2, leukotriene B(4), or nitric oxide) in the biological sample.

The invention also features a method for generating an animal having reduced body fat as compared to a corresponding control animal. The method can involve administering to the animal an agent effective to alter TLR4, MD-2, or CD14 activity in the animal.

In yet another aspect, the invention features a transgenic non-human animal, the nucleated cells of which contain a transgene, wherein the presence of the transgene results in altered TLR4, MD-2, or CD14 activity as compared to the TLR4, MD-2, or CD14 activity of a corresponding control animal. The transgenic non-human animal can have decreased body fat as compared to the control animal. The transgene can include a TLR4, MD-2, or CD14 nucleic acid sequence containing a variant. The transgene may or may not be integrated into the corresponding endogenous TLR4, MD-2, or CD14 locus of the transgenic non-human animal. The transgenic non-human animal can have increased bone density as compared to the control animal. The transgenic non-human animal can be bovine, ovine, porcine, or fowl.

In another aspect, the invention features a population of animals having reduced TLR4, MD-2, or CD14 activity as compared to a corresponding population of control animals, wherein the animals have decreased body fat as compared to the corresponding control animals. The population of animals can be bovine, ovine, porcine, or fowl. The animals within the population can contain a TLR4, MD-2, or CD14 nucleic acid, the nucleotide sequence of which includes a variant. The population of animals can be homozygous or heterozygous for the variant. The animals within the population can contain a TLR4, MD-2, or CD14 polypeptide, the amino acid sequence of which includes a variant.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

In general, the invention provides materials and methods for generating or identifying an animal having reduced body fat and increased bone density. In particular, the invention provides methods for generating an animal having altered levels of TLR4, MD-2, and CD14 activity (e.g., by treatment with one or more agents that affect TLR4, MD-2, or CD14 activity), and methods for identifying an animal as having altered levels of TLR4, MD-2, and/or CD14 activity. Altered TLR4, MD-2, and/or CD14 activity may result from one or more amino acid sequence variants within a TLR4, MD-2, or CD14 polypeptide; such amino acid sequence variants typically result from nucleotide sequence variants within the nucleic acids encoding these polypeptides. As such, the invention further provides methods for generating or identifying animals containing TLR4, MD-2, and/or CD14 nucleotide or polypeptide sequence variants.

TLR4 is the main receptor that transduces signals delivered by endotoxin (lipopolysaccharide (LPS)) and other bacterial products. TLR4 is expressed as a homodimer on the surface of adipocytes and osteoblasts and their common precursor, the stromal cell. TLR4 also is expressed on macrophages, dendritic cells, and osteoclasts and their common precursor in the bone marrow. TLR4 does not have high affinity for LPS, and other molecules (e.g., MD-2 and CD14) are required to facilitate interaction of LPS with TLR4.

MD-2 is a 25-kDa secreted protein that associates non-covalently with the extracellular domain of TLR4 (Shimazu et al., (1999) J. Exp. Med. 189:1777-1782). MD-2 is believed to stabilize the receptor complex and to facilitate localization of TLR4 at the plasma membrane in macrophages. Furthermore, MD-2 is required for TLR4 recognition of LPS. MD-2-associated TLR4 homodimers do not bind LPS directly, however. LPS first binds to a soluble LPS binding protein (LBP), and LBP then can bind to CD14.

CD14 is a glycosylphosphatidylinositol- (GPI-) linked protein that is expressed strongly on the surface of monocytes and weakly on the surface of granulocytes. CD14 also is expressed by most tissue macrophages. In mice stimulated with LPS, CD14 expression also was detected in non-myeloid cell types (e.g., hepatocytes and several epithelial cell types; Fearns et al. (1995) J. Exp. Med. 181:857-866). In addition, soluble forms of CD14 have been detected in serum and tissue culture supernatants of cells transfected with CD14 expression constructs.

Interaction between LPS and LBP facilitates the binding of LPS to CD14 (Hailman et al. (1994) J. Exp. Med. 179:269-277). LBP/LPS can bind to either soluble or GPI-linked CD 14. While the exact mechanism is still unclear, it is thought that LBP transfers LPS to CD14, thereby activating TLR4. See, Janeway and Medzhitov (2002) Ann. Rev. Immunol. 20:197; Barton and Medzhitov (2002) Curr. Top. Microbiol. Immunol. 270:81; Medzhitov (2001) Nat. Rev. Immunol. 1:135; Heine and Lein (2003) Int. Arch. Allergy Immunol. 130:180; Modlin (2002) Ann. Allergy Asthma Immunol. 88:543; and Dunne and O'Neill (2003) Sci. STKE 2003:re3.

In the absence of infection, TLR4 plays a role in normal development or homeostasis of body fat and/or bone density. An alteration (e.g., a reduction) in activity of TLR4 or other polypeptides in the TLR4 signaling pathway (e.g., MD-2 or CD14) can result in reduced body fat and increased bone density. See, e.g., the Examples below, and U.S. Provisional Application Ser. No. 60/478,067, which is incorporated herein by reference in its entirety. Consequently, agents that alter (e.g., inhibit) the activity of TLR4, MD-2, CD 14, or other polypeptide in the pathway can be used to reduce fat mass in an animal, thus increasing the relative lean mass. Such agents also can be used to increase bone density in an animal. Alternatively, animals naturally having altered TLR4, MD-2, or CD14 activity (e.g., animals containing variant TLR4, MD-2, or CD14 polypeptides) can be bred to produce offspring having altered activity. Animals also can be genetically engineered to have altered TLR4, MD-2, or CD14 activity, with a concomitant increase in lean to fat body mass. Such animals may be commercially valuable, particularly if they are destined for human consumption. Since such animals also may have increased bone density, they can be characterized by increased bone strength and resistance to fracture. These animals therefore may be better able to withstand transport.

TLR4, MD-2, and CD14 Nucleic Acids

TLR4, MD-2, and CD14 activity can be affected by the presence of variant TLR4, MD-2 and CD14 nucleic acids and polypeptides. The term “nucleic acid” as used herein encompasses both RNA and DNA, including genomic DNA. A nucleic acid can be double-stranded or single-stranded (e.g., a sense single strand or an anti-sense single strand). As used herein, a nucleotide sequence “variant” refers to any alteration in a TLR4, MD-2, or CD14 reference nucleotide sequence. A reference nucleotide sequence also can be referred to as a “wild type” sequence. Nucleotide sequence variants include variations that occur in coding and non-coding regions, including exons, introns, and untranslated sequences. Nucleotide sequence variants can include single nucleotide substitutions, deletions of one or more nucleotides, and insertions of one or more nucleotides.

TLR4, MD-2, and CD14 reference sequences can be identified using standard molecular techniques (e.g., standard cloning, amplification, and sequencing techniques). For example, a reference sequence can be identified by (1) obtaining a biological sample from a control animal (e.g., an animal having normal levels of TLR4, MD-2, and CD14 activity), (2) isolating nucleic acids from the sample, (3) amplifying at least a portion of a TLR4, MD-2, or CD14 nucleic acid (e.g., using PCR), and (4) sequencing the amplification product. Alternatively, a reference sequence (e.g., a TLR4 reference sequence) can be a consensus nucleotide sequence identified by aligning and comparing the TLR4 nucleotide sequences present in a plurality of animals.

In addition, a reference TLR4 nucleotide sequence can include any known TLR4 nucleic acid sequence from a particular species. Examples of known TLR4 sequences that can be found in GenBank® include those having the following Accession Nos. (listed by species): cow (Bos taurus; NM_(—)174198, AB056444, and AF310952); pig (Sus scrofa; AY289532); chicken (Gallus gallus; AY064697); horse (Equus caballus; AY005808); dog (Canis familiaris; AB080363); cat (Felis catus; BAB43947); human (Homo sapiens; NM_(—)138557, NM_(—)138556, NM_(—)138554, NM_(—)003266, AF177765, AF172171, AF172170, AF172169, AH009665, AF177766, and U88880); mouse (Mus musculus; NM_(—)021297, AL805946, AF177767, AF222309, AF185285, AF110133, and AF095353); rat (Rattus norvegicus; NM_(—)019178); Chinese hamster (Cricetulus griseus; AF153676); gorilla (Gorilla gorilla; AF497565, AF497564, AF497563, and AH011592); orangutan (Pongo pygmaeus; AF497562, AF497561, AF497560, and AH011591); olive baboon (Papio hamadryas anubis; AH008378, AF180964, AF180963, and AF180962); pygmy chimpanzee (Pan paniscus; AH008351, AF179220, AF179219, and AF179218); and rhesus monkey (Macaca mulatta; AF162474).

Similarly, a reference MD-2 or CD14 nucleotide sequence can include any known MD-2 or CD14 nucleic acid sequence from a particular species. MD-2 (also known as LY96) sequences can be found in GenBank® for the following species: cow (B. taurus; AF368418, NM_(—)174111, and AF368418); chicken (G. gallus; BI066409); horse (E. caballus; AF200416); sheep (Ovis aries; AY289201 and AJ535322); human (H. sapiens; NM_(—)015364, BC020690, and AB018549); mouse (M. musculus; NM_(—)016923 and AB018550); Chinese hamster (C. griseus; AF325501); and rabbit (Oryctolagus cuniculus; AY101395). CD14 sequences also can be found in GenBank® for the following species: cow (B. taurus; AF141313, D84509, and U48356); human (H. sapiens; BC010507, NM_(—)000591, BT007331, AJ491310, AY044269, X06882, AF097335, AF097942, X13334, X74984, M86511, and U00699); mouse (M. musculus; NM_(—)009841, D10912, AB039063, AB039062, AB039061, AB039060, AB039059, AB039058, AB039057, AB039056, AB039055, AB039054, X13987, and X13333); rat (Rattus norvegicus; NM_(—)021744, U51804, AF087944, and AF087943); and rabbit (O. cuniculus; D16545, M90488, and M85233).

In some embodiments, a variant TLR4, MD-2, or CD14 nucleic acid can encode a variant TLR4, MD-2, or CD14 polypeptide that contains an amino acid sequence variant. The term “polypeptide” refers to any chain of at least four amino acid residues (e.g., 4-8, 9-12, 13-15, 16-18, 19-21, 22-100, 100-150, 150-200, 200-300 residues, or a full-length TLR4, MD-2, or CD14 polypeptide), regardless of post-translational modification (e.g., phosphorylation or glycosylation).

An amino acid sequence “variant” is any alteration (e.g., substitution, deletion, or insertion) in a TLR4, MD-2, or CD14 reference amino acid sequence. Amino acid substitutions may be conservative or non-conservative. Conservative amino acid substitutions replace an amino acid with an amino acid of the same class, whereas non-conservative amino acid substitutions replace an amino acid with an amino acid of a different class. Conservative amino acid substitutions typically have little effect on the structure or function of a polypeptide. Examples of conservative substitutions include, without limitation, amino acid substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.

Non-conservative substitutions may result in a substantial change in the hydrophobicity of the polypeptide or in the bulk of a residue side chain. In addition, non-conservative substitutions may make a substantial change in the charge of the polypeptide, such as reducing electropositive charges or introducing electronegative charges. Examples of non-conservative substitutions include a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.

Variant TLR4 polypeptides may or may not have LPS-binding and/or gene expression-enhancing activity, or may have activity that is altered relative to the reference polypeptide. Certain nucleotide sequence variants do not alter the encoded amino acid sequence. Such variants, however, may alter regulation of transcription as well as mRNA stability. For example, nucleotide sequence variants can occur in intron sequences or in 5′ or 3′ untranslated sequences. Nucleotide sequence variants that do not change the amino acid sequence also can be within an exon.

Variant mouse, bovine, and human TLR4 and CD14 nucleic acids and polypeptides have been identified. See, Smirnova et al. (2000) Genome Biol. 1:RESEARCH002; White et al. (2003) Proc. Natl. Acad. Sci. USA 100:10364-10369; Smirnova et al. (2001) Genetics 158:1657-1664, Unkelbach et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19:932-938; and Hayden et al. (2000) Hum. Mutat. 15:122. In particular, studies of TLR4 amino acid sequences in humans revealed both common and rare polymorphisms. These TLR4 amino acid sequence variants, including, without limitation, substitution of glycine for aspartic acid at residue 299 (Asp299Gly) and substitution of isoleucine for threonine at residue 399 (Thr399Ile), may reduce the activity of TLR4.

Methods for Identifying Animals Having Reduced Body Fat and Increased Bone Density

The invention provides methods for identifying an animal (a cow, pig, horse, goat, sheep, chicken, turkey, dog, cat, bird, monkey, rat, mouse, or fish) as having reduced body fat and/or increased bone density, based on an altered level of TLR4, MD-2, or CD14 activity in a biological sample obtained from the animal. As used herein, “biological sample” refers to any sample obtained, directly or indirectly, from a subject animal or a control animal. Representative biological samples that can be obtained from an animal include or are derived from biological tissues, biological fluids, and biological elimination products (e.g., feces). Biological tissues can include biopsy samples or swabs of the biological tissue of interest, e.g., nasal swabs, throat swabs, or dermal swabs. The tissue of interest to sample (e.g., by biopsy or swab) can be, for example, an eye, a tongue, a cheek, a hoof, a beak, a snout, a foot, a hand, a mouth, a teat, the gastrointestinal tract, a feather, an ear, a nose, a mucous membrane, a scale, a shell, the fur, or the skin.

Biological fluids can include bodily fluids (e.g., urine, milk, lachrymal fluid, vitreous fluid, sputum, cerebrospinal fluid, sweat, lymph, saliva, semen, blood, or serum or plasma derived from blood); a lavage such as a breast duct lavage, lung lavage, gastric lavage, rectal or colonic lavage, or vaginal lavage; an aspirate such as a nipple or teat aspirate; a fluid such as a cell culture or a supernatant from a cell culture; and a fluid such as a buffer that has been used to obtain or resuspend a sample, e.g., to wash or to wet a swab in a swab sampling procedure. Biological samples can be obtained from an animal using methods and techniques known in the art. See, for example, Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds.), 1993, American Society for Microbiology, Washington D.C.).

As used herein, “altered” TLR4, MD-2, and CD14 activity encompasses both increased and reduced activity as compared to a control level of activity. Typically, reduced TLR4, MD-2, or CD14 activity in an animal can be correlated with reduced body fat and increased bone density. By “reduced” TLR4, MD-2, or CD14 activity is meant any decrease in activity as compared to a control level of activity. For example, the level of TLR4, MD-2, or CD14 can be decreased between 5% and 100% (e.g., 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, or 100%) relative to a control level of TLR4, MD-2, or CD14 activity.

The term “control level” of TLR4, MD-2, or CD14 activity can refer to a standard or average level of activity. In one embodiment, for example, a control level of TLR4 activity can be the level of activity in a biological sample obtained from a corresponding control animal that contains a reference TLR4 nucleic acid sequence as described above. Typically, a “corresponding control animal” is an animal of the same species as the subject animal in which TLR4, MD-2, or CD14 activity is to be evaluated. In another embodiment, for example, a control level of TLR4 activity can be an average level of TLR4 activity as determined from assays of biological samples obtained from a plurality of corresponding control animals. In still another embodiment, a control level of TLR4 activity can be a standard level of TLR4 activity obtained with a particular amount of TLR4.

LPS stimulation through TLR4, MD-2, and CD14 activates the expression of numerous genes. Thus, the level of TLR4, MD-2, or CD14 activity in an animal or a cell can be evaluated by measuring expression of any of these genes. Genes activated by LPS through TLR4, MD-2, and CD14 include, for example, cytokines such as interleukins or interleukin receptors (e.g., IL-1R, IL-1β, IL-4, IL-6, IL-6R, IL-7, IL-8, IL-10, IL-11, IL-12), tumor necrosis factor α or β (TNFα or β), osteoclast differentiation factor (ODF), leptin, chemokines such as inducible protein 10 (IP-10), macrophage inflammatory protein 1α (MIP-1α), monocyte chemoattractant protein 1 (MCP-1), CC chemokine ligand 2 (CCL2), CC chemokine receptor, CXC chemokine LIX, and CC chemokine MIP-3α.

Other genes that are activated by TLR4 include cylooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), extracellular signal-regulated kinase 1 (ERK1), ERK2, IL-1 receptor-associated kinase (IRAK), nuclear factor-kappaB (NF-κB), activating protein-1 (AP-1), TLR2, secretory IL-1 receptor antagonist (sIL-1Ra), insulin-like growth factor binding protein-3 (IGFBP-3), vascular cell adhesion protein 1 (VCAM-1), p-selectin, β-integrin, vascular endothelial growth factor, β-nerve growth factor (NGF), lymphotoxin R, interferon regulatory factor 1 (IRF-1), mitochondrial hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase), aldehyde dehydrogenase 2, neurotensin receptor 2, and protooncogenes such as c-Fos, Fos-B, Fra-2, Jun-B, Jun-D, or Egr-1. Surface markers that are expressed when TLR4 is activated include CD40, CD80, CD86, MHC class I, MHC class II, and CD25. Expression of any of these genes can be measured to evaluate TLR4, MD-2, or CD14 activity.

Expression of genes that are activated by TLR4, MD-2, and CD 14, including those listed herein, can be monitored by assessing mRNA or protein levels using standard molecular biology techniques, for example. Western blotting or immunoassays (e.g., ELISA) can be used to monitor protein production. Northern blotting, gene chip arrays, or polymerase chain reaction (PCR) techniques can be used to assess mRNA production. PCR refers to a procedure or technique in which target nucleic acids are enzymatically amplified. Sequence information from the ends of the region of interest or beyond typically is employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase (RT) can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12(9):1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292.

Other examples of genes activated by TLR4 include members of intracellular signaling pathways such as NF-κB, AP1, and MAP (mitogen-activated protein) kinases (ERK, p38, JNK); Akt and phosphatidylinositol-3′-kinase (PI-3-K); protein kinase C; signal transducer and activator of transcription 1α (STAT 1α) and STAT1β; p38 (stress-activated protein kinase); Tollip; and c-Jun Kinase. TLR4-stimulated activation of these pathways can be easily monitored using immunoblot or flow cytometric analysis with activation-state-specific antibodies directed against components of the monitored biochemical pathway.

In addition, any other suitable method can be used to determine whether an animal has altered TLR4, MD-2, or CD14 activity. For example, small molecules such as PGE2 (prostaglandin E2), leukotriene B(4), and nitric oxide (NO) typically are synthesized when TLR4 is activated. These end products can be detected using sandwich ELISA techniques or by colorometric chemical reactivity assays. Furthermore, assays that include detection of protein-protein interactions can be used to assess the level of TLR4, MD-2, or CD14 activity. See, for example, Fotin-Mleczek et al. (2000) Biotechniques 31:22-26, which describes a green fluorescent protein-based mammalian two-hybrid system for detecting protein-protein interactions.

Alternatively, the activity of TLR4, MD-2, and/or CD14 can be measured indirectly by assessing the level of TLR4, MD-2, and CD14, since low or no expression of these proteins may indicate reduced activity. TLR4, MD-2, and CD14 levels can be measured in serum, for example, since (a) TLR4 may exist in a soluble form, (b) MD-2 is secreted when not bound to TLR4, and (c) CD14 is expressed in part as a soluble form. High levels of these soluble proteins (especially MD-2 and TLR4) may inhibit TLR4 function. Alternatively, TLR4, MD-2, and CD14 protein levels also can be measured by tissue staining using immunohistochemistry techniques known in the art, for example.

As an alternative to examining effects on gene expression, the activity of TLR4, MD-2, and CD14 can be evaluated by determining whether or not an animal contains a TLR4, MD-2, or CD14 nucleotide sequence variant. For example, TLR4, MD-2, and CD14 nucleotide sequence variants can be detected by, for example, sequencing exons, introns, 5′ untranslated sequences, or 3′ untranslated sequences, by performing allele-specific hybridization, allele-specific restriction digests, mutation specific PCR (MSPCR), real-time PCR (Heesen et al. (2003) Clin. Chim. Acta. 333:47-49), single-stranded conformational variant (SSCP) detection (Schafer et al. (1995) Nat. Biotechnol. 15:33-39), denaturing high performance liquid chromatography (DHPLC, Underhill et al. (1997) Genome Res. 7:996-1005), infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry (WO 99/57318), and combinations of such methods (e.g., as described in Schmitt et al., supra).

Genomic DNA typically is used in the analysis of nucleotide sequence variants. Genomic DNA can be extracted from a biological sample such as a peripheral blood sample, but also can be extracted from other biological samples, including tissues (e.g., mucosal scrapings of the lining of the mouth or from renal or hepatic tissue). Standard methods can be used to extract genomic DNA from a blood or tissue sample, including, for example, phenol extraction. Alternatively, genomic DNA can be extracted with kits such as the QIAamp® Tissue Kit (Qiagen, Valencia, Calif.), Wizard® Genomic DNA purification kit (Promega, Madison, Wis.) and the A.S.A.P.™ Genomic DNA isolation kit (Boehringer Mannheim, Indianapolis, Ind.).

Typically, an amplification step is performed before proceeding with the detection method. For example, exons or introns of a TLR4 gene can be amplified and then directly sequenced using standard techniques. Dye primer sequencing can be used to increase the accuracy of detecting heterozygous samples.

Allele specific hybridization also can be used to detect nucleotide sequence variants, including complete haplotypes of a mammal. See, Stoneking et al. (1991) Am. J. Hum. Genet. 48:370-382; and Prince et al. (2001) Genome Res. 11:152-162. In practice, samples of DNA or RNA from one or more animals can be amplified using pairs of primers and the resulting amplification products can be immobilized on a substrate (e.g., in discrete regions). Hybridization conditions can be selected such that a nucleic acid probe can specifically bind to the sequence of interest, e.g., a TLR4 nucleic acid molecule containing a particular nucleotide sequence variant. Such hybridizations typically are performed under high stringency, as some nucleotide sequence variants include only a single nucleotide difference. High stringency conditions can include, for example, the use of low ionic strength solutions and high temperatures for washing. For example, nucleic acid molecules can be hybridized at 42° C. in 2×SSC (0.3M NaCl/0.03 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) and washed in 0.1×SSC (0.015M NaCl/0.0015 M sodium citrate), 0.1% SDS at 65° C. Hybridization conditions can be adjusted to account for unique features of the nucleic acid molecule, including length and sequence composition. Probes can be labeled (e.g., fluorescently) to facilitate detection. In some embodiments, one of the primers used in the amplification reaction is biotinylated (e.g., 5′ end of reverse primer) and the resulting biotinylated amplification product is immobilized on an avidin or streptavidin coated substrate.

Allele-specific restriction digests can be performed in the following manner. For nucleotide sequence variants that introduce a restriction site, restriction digestion with the particular restriction enzyme can differentiate the alleles. For nucleotide sequence variants that do not alter a common restriction site, mutagenic primers can be designed that introduce a restriction site when the variant allele is present or when the wild type allele is present. A portion of a nucleic acid can be amplified using the mutagenic primer and a wild type primer, followed by digest with the appropriate restriction endonuclease.

Certain variants, such as insertions or deletions of one or more nucleotides, can change the size of the DNA fragment encompassing the variant. The insertion or deletion of nucleotides can be assessed by amplifying the region encompassing the variant and determining the size of the amplified products in comparison with size standards. For example, a region of a TLR4 nucleic acid can be amplified using a primer set from either side of the variant. One of the primers typically is labeled with, for example, a fluorescent moiety to facilitate sizing. The amplified products can be electrophoresed through acrylamide gels with a set of size standards that are labeled with a fluorescent moiety that differs from the primer.

PCR conditions and primers can be developed that amplify a product only when the variant allele is present or only when the wild type allele is present (MSPCR or allele-specific PCR). For example, a sample DNA and a control DNA can be amplified separately using either a wild type primer or a primer specific for the variant allele. Each set of reactions is then examined for the presence of amplification products using standard methods to visualize the DNA. The reactions can be electrophoresed through an agarose gel and the DNA visualized by staining with ethidium bromide or other DNA intercalating dye. In a DNA sample from a heterozygous animal, reaction products would be detected in each reaction. Samples containing solely the wild type allele would have amplification products only in the reaction using the wild type primer. Similarly, samples containing solely the variant allele would have amplification products only in the reaction using the variant primer. Allele-specific PCR also can be performed using allele-specific primers that introduce priming sites for two universal energy-transfer-labeled primers (e.g., one primer labeled with a green dye such as fluoroscein and one primer labeled with a red dye such as sulforhodamine). Amplification products can be analyzed for green and red fluorescence in a plate reader. See, Myakishev et al. (2001) Genome 11:163-169.

Mismatch cleavage methods also can be used to detect differing sequences by PCR amplification, followed by hybridization with the wild type sequence and cleavage at points of mismatch. Chemical reagents, such as carbodiimide or hydroxylamine and osmium tetroxide can be used to modify mismatched nucleotides to facilitate cleavage.

Alternatively, the activity of TLR4, MD-2, and CD14 can be evaluated by determining whether or not an animal contains a TLR4, MD-2, or CD14 amino acid sequence variant. A TLR4, MD-2, and CD14 polypeptide containing one or more amino acid sequence variants can be detected using, for example, antibodies that have specific binding affinity for the particular variant polypeptide. Variant polypeptides can be produced in various ways, including recombinantly, for example. Host animals such as rabbits, chickens, mice, guinea pigs and rats can be immunized by injection of a particular variant polypeptide. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Polyclonal antibodies are heterogenous populations of antibody molecules that are contained in the sera of the immunized animals. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be prepared using a variant polypeptide and standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler et al. (1975) Nature 256:495, the human B-cell hybridoma technique (Kosbor et al. (1983) Immunology Today 4:72; Cote et al. (1983) Proc. Natl. Acad. Sci USA 80:2026), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibodies of the invention can be cultivated in vitro and in vivo.

Antibody fragments that have specific binding affinity for a particular variant polypeptide can be generated by known techniques. For example, such fragments include but are not limited to F(ab′)2 fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab′)2 fragments. Alternatively, Fab expression libraries can be constructed. See, for example, Huse et al., Science, 246:1275 (1989). Once produced, antibodies or fragments thereof can be tested for recognition of variant TLR4, MD-2, or CD14 polypeptides using standard immunoassay methods including ELISA techniques, radioimmunoassays and Western blotting. See, Short Protocols in Molecular Biology, Chapter 11, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al., 1992.

Methods for Generating Animals Having Reduced Body Fat and Increased Bone Density

The invention provides methods for generating animals having reduced body fat and increased bone density. In addition, the invention provides animals generated using the methods disclosed herein, as well as progeny and cells of such animals. Suitable animals include, for example, farm animals such as cattle, pigs, sheep, goats, horses, and poultry (e.g., chickens and turkeys), and rodents such as rats, guinea pigs, and mice. Such animals can be generated by (1) cross-breeding animals identified as containing one or more variant TLR4, MD-2, or CD14 nucleic acids or polypeptides; (2) generating non-human animals that contain a transgene, the presence of which results in altered TLR4, MD-2, or CD14 activity; or (3) treating animals with one or more agents that affect TLR4, MD-2, and/or CD14 activity.

In one embodiment, animals identified as having reduced body fat and/or increased bone density based on altered levels of TLR4, MD-2, or CD14 activity, as described above, can be bred to generate offspring having altered TLR4, MD-2, or CD14 activity. For example, a first animal identified as having a TLR4, MD-2, or CD14 nucleotide or amino acid sequence variant can be bred with a second animal identified as having a TLR4, MD-2, or CD14 nucleotide or amino acid sequence variant. The first and second animals may contain the same sequence variants, or the sequence variants of the first and second animals may differ from each other. Further, the first and second animals may be identified as having a single TLR4, MD-2, or CD14 sequence variant, or one or both animals can have multiple TLR4, MD-2, and/or CD14 sequence variants. The offspring can be evaluated to determine whether they also have altered levels of TLR4, MD-2, or CD14 activity, and then can be bred to produce further generations. Using such methods, a population of animals can be generated that has reduced body fat and/or increased bone density.

In another embodiment, non-human animals can be generated that contain a transgene, the presence of which results in altered (e.g., reduced) TLR4, MD-2, or CD14 activity. As used herein, the term “transgenic non-human animal” includes the founder transgenic non-human animals as well as progeny of the founders, and tissues and cells (e.g., adipocytes or myocytes) obtained from the transgenic non-human animals.

The nucleated cells of transgenic non-human animals contain a transgene that may include a TLR4, MD-2, or CD14 nucleotide sequence, such as a variant TLR4, MD-2, or CD14 nucleotide sequence that encodes a TLR4, MD-2, or CD14 polypeptide having altered activity. Alternatively, a transgene can include a nucleotide sequence that is unrelated to TLR4, MD-2, or CD14. For example, a transgene can include a non-TLR4, non-MD-2, or non-CD 14 nucleotide sequence (e.g., a selectable marker as described below), and can be targeted to an endogenous TLR4, MD-2, or CD14 nucleotide sequence to prevent expression of a functional gene product. As another alternative, a transgene can contain a nucleotide sequence that is targeted to or encodes a non-TLR4, non-MD-2, or non-CD14 polypeptide (e.g., LBP, MD-1, or RP105) that affects TLR4, MD-2, or CD14 activity.

Transgenic non-human animals can be generated to contain a randomly integrated transgene that includes a variant TLR4, MD-2, or CD14 nucleic acid sequence, while the endogenous TLR4, MD-2, and CD14 nucleic acid sequences remain. In such animals, expression of the TLR4, MD-2, or CD14 sequence in the transgene can be under the control of a constitutive or regulated promoter, for example. Alternatively, an endogenous TLR4, MD-2, or CD14 nucleic acid can be replaced through homologous recombination with a transgene containing a variant TLR4, MD-2, or CD14 nucleic acid. As another alternative, a “knockout” transgenic non-human animal can be generated using homologous recombination to replace an endogenous TLR4, MD-2, or CD14 nucleic acid with a transgene containing an unrelated sequence, such that expression of a functional TLR4, MD-2, or CD14 gene product from the endogenous sequence is not detectable. In still another alternative, a transgene that includes a variant TLR4, MD-2, or CD 14 nucleic acid can be randomly integrated into a knockout non-human animal, thus producing a “knock in” animal. See, Shastry (1998) Mol. Cell Biochem. 181:163-179, for a review of gene targeting technology.

A transgene can include additional regulatory elements, including for example, promoters, inducible elements, or other upstream promoter elements, operably linked to a nucleic acid sequence encoding a polypeptide (e.g., a TLR4 polypeptide). As used herein, “operably linked” refers to positioning of a regulatory element in a transgene relative to the nucleic acid sequence encoding the polypeptide in such a way as to permit or facilitate expression of the encoded polypeptide. For example, a tissue specific promoter can be operably linked to a variant TLR4, MD-2, or CD14 nucleic acid sequence within a transgene. Alternatively, a promoter that facilitates the expression of a nucleic acid molecule without significant tissue- or temporal-specificity can be used.

In some embodiments, a transgene includes a tag sequence that encodes a “tag” that is designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., localization or easy detection). For example, a tag sequence can be inserted in the nucleic acid sequence encoding a variant TLR4, MD-2, or CD14 polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include green fluorescent protein (GFP), glutathione S-transferase (GST), and FLAGTM tag (Kodak, New Haven, Conn.), which has the amino acid sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:1).

A transgene used to produce a knockout non-human animal can include, for example, a nucleic acid sequence encoding a selectable marker. Typically, the selectable marker is flanked by sequences homologous to the sequences flanking the desired insertion site, such that the gene of interest is targeted and replaced via homologous recombination. It is not necessary for the flanking sequences to be immediately adjacent to the desired insertion site. Suitable markers for positive drug selection include, for example, the aminoglycoside 3N phosphotransferase gene that imparts resistance to geneticin (G418, an aminoglycoside antibiotic), and other antibiotic resistance markers, such as the hygromycin-B-phosphotransferase gene that imparts hygromycin resistance. Other selection systems can include negative-selection markers such as the thymidine kinase (TK) gene from herpes simplex virus. Constructs utilizing both positive and negative drug selection also can be used. For example, a construct can contain the aminoglycoside phosphotransferase gene and the TK gene. In this system, cells are selected that are resistant to G418 and sensitive to gancyclovir.

Various techniques known in the art can be used to introduce transgenes into non-human animals to produce founder lines in which the transgene is integrated into the genome. To create non-human animals containing a transgene in all cells, it is necessary to introduce a transgene construct into the germ cells (sperm or eggs, i.e., the “germ line”) of the desired species. Genes or other DNA sequences can be introduced into the pronuclei of fertilized eggs by microinjection. Following pronuclear fusion, the developing embryo may carry the introduced gene in all its somatic and germ cells since the zygote is the mitotic progenitor of all cells in the embryo. When targeting an endogenous sequence, it typically is desirable to generate and screen a large number of animals since targeted insertion of a transgene is a relatively rare event. Because of this, it can be advantageous to work with the large cell populations and selection criteria that are characteristic of cultured cell systems. However, for production of non-human transgenic animals from an initial population of cultured cells, it is necessary that a cultured cell containing the desired transgene construct be capable of generating a whole animal. This generally is accomplished by placing the cell into a developing embryo environment of some sort.

Cells capable of giving rise to at least several differentiated cell types are “pluripotent.” Pluripotent cells capable of giving rise to all cell types of an embryo, including germ cells, are hereinafter termed “totipotent” cells. Totipotent murine cell lines (embryonic stem, or “ES” cells) have been isolated by culture of cells derived from very young embryos (blastocysts). Such cells are capable, upon incorporation into an embryo, of differentiating into all cell types, including germ cells. As such, these cells can be employed to generate animals lacking an endogenous TLR4, MD-2, or CD14 nucleic acid, for example. That is, cultured ES cells can be transformed with a transgene construct and cells selected in which the endogenous TLR4, MD-2, or CD14 gene is inactivated or replaced. Nucleic acid constructs can be introduced into ES cells using, for example, electroporation or any other standard technique. Selected cells can be screened for gene targeting events. For example, PCR can be used to confirm the presence of the transgene.

The ES cells further can be characterized to determine the number of targeting events. For example, genomic DNA can be harvested from ES cells and used for Southern analysis. See, for example, Sections 9.37-9.52 of Sambrook et al., Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; N.Y., 1989.

To generate a transgenic (e.g., knockout) animal, ES cells having at least one successfully targeted (i.e., transgenic) TLR4, MD-2, or CD14 allele can be incorporated into a developing embryo. This can be accomplished through injection into the blastocyst cavity of a murine blastocyst-stage embryo, by injection into a morula-stage embryo, by co-culture of ES cells with a morula-stage embryo, or through fusion of the ES cell with an enucleated zygote. The resulting embryo is raised to sexual maturity and bred in order to obtain animals whose cells (including germ cells) carry the transgenic TLR4, MD-2, or CD14 allele. If the original ES cell was heterozygous for the transgenic TLR4, MD-2, or CD14 allele, several of these animals can be bred with each other in order to generate animals homozygous for the inactivated allele.

Alternatively, direct microinjection of DNA into eggs can be used to avoid the manipulations required to generate an animal from a cultured cell. Fertilized eggs are “totipotent,” i.e., capable of developing into an adult without further substantive manipulation other than implantation into a surrogate mother. To enhance the probability of homologous recombination when eggs are directly injected with transgene constructs, it is useful to incorporate at least about 8 kb of homologous DNA into the targeting construct. In addition, it is also useful to prepare the knockout constructs from isogenic DNA.

Embryos derived from microinjected eggs can be screened for homologous recombination events in several ways. For example, if a TLR4, MD-2, or CD14 gene is interrupted by a coding region that produces a detectable (e.g., fluorescent) gene product, then the injected eggs can be cultured to the blastocyst stage and analyzed for presence of the detectable gene product. Embryos with fluorescing cells, for example, are then implanted into a surrogate mother and allowed to develop to term. Alternatively, injected eggs are allowed to develop and DNA from the resulting pups analyzed by PCR or RT-PCR for evidence of homologous recombination.

Nuclear transplantation also can be used to generate non-human animals of the invention. For example, fetal fibroblasts can be genetically modified such that they contain an inactivated endogenous TLR4, MD-2, or CD14 gene and/or express a variant TLR4, MD-2, or CD14 nucleic acid, and then fused with enucleated oocytes. After activation of the oocytes, the eggs are cultured to the blastocyst stage, and implanted into a recipient. See, Cibelli et al. (1998) Science 280:1256-1258. Adult somatic cells including, for example, cumulus cells and mammary cells, can be used to produce animals such as mice and sheep, respectively. See, for example, Wakayama et al. (1998) Nature 394:369-374; and Wilmut et al. (1997) Nature 385:810-813. Nuclei can be removed from genetically modified adult somatic cells and transplanted into enucleated oocytes. After activation, the eggs can be cultured to the 2-8 cell stage, or to the blastocyst stage, and implanted into a suitable recipient. Wakayama et al., 1998, supra.

Once transgenic non-human animals have been generated, expression of an encoded polypeptide (e.g., a variant TLR4 polypeptide) can be assessed using standard techniques. Initial screening can be accomplished by Southern blot analysis to determine whether or not integration of the transgene has taken place. See, for example, sections 9.37-9.52 of Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; N.Y., for a description of Southern analysis. PCR techniques also can be used in the initial screening. Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplified. See, for example, Lewis (1992) Genet. Eng. News 12(9):1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292.

Expression of a nucleic acid sequence encoding, for example, a variant TLR4, MD-2, or CD14 polypeptide in the tissues of a transgenic non-human animal can be assessed using techniques that include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal (e.g., adipose or muscle tissue), in situ hybridization analysis, Western analysis, immunoassays such as enzyme-linked immunosorbent assays, and reverse-transcriptase PCR (RT-PCR). Standard breeding techniques can be used to create animals homozygous for the transgene from the initial heterozygous founder animals. Homozygosity may not be required, however, as the phenotype may be observed in hemizygotic animals.

In another embodiment, non-human animals having reduced body fat and increased bone density can be generated by treatment with one or more agents that alter (e.g., reduce) TLR4, MD-2, and/or CD14 activity. Such agents can be identified using in vitro or in vivo methods, or combinations of in vitro and in vivo methods. For example, a compound that decreases fat mass or increases bone density can be identified by contacting a cell in vitro with a test compound in the presence of an agonist (e.g., lipid A, or mono or disaccharides such as those disclosed in U.S. Patent Publication 20020077304), and then monitoring TLR4, MD-2, or CD14 activity. In some embodiments, cells can be obtained from a particular subject to be tested. After in vitro testing, compounds that inhibit TLR activity then can be administered to a non-human animal. Alternatively, test compounds can be directly administered to a non-human animal without initial testing in vitro. Cells that can be used in such methods include, for example, any type of cell line that expresses some form of TLR4, or that can be transfected to express TLR4. These can include, without limitation, human embryonic kidney (e.g., HEK293 cells), adipocyte cell lines, B-cells and B-cell lines, other macrophage cell lines (e.g., RAW), neutrophils, peripheral blood leukocytes, cells cultured from blood or bone marrow (e.g., dendritic cells), and primary cell cultures. In addition, compounds can be identified using cell lines that have been stably or transiently transfected to express components of the TLR4 signaling pathway (e.g., TLR4, MD-2, CD14, or intracellular components such as MyD88). Cells transfected to express particular polypeptides associated with TRL4 signaling can be useful to determine whether an agonist or antagonist requires all or only some of the signaling components.

Suitable test compounds can affect TLR4, MD-2, or CD14 directly or indirectly (e.g., by inhibiting an upstream molecule), and can include, for example, small molecules, an extracellular matrix (ECM) preparation, glycosaminoglycans, glycoproteins, polysaccharides, polypeptides, and nucleic acids (e.g., polymerized nucleic acids). The glycoprotein can include hyaluronic acid or a hyaluronic acid-protein conjugate, heparan sulfate or a heparan sulfate protein conjugate, or chondroitin sulfate. Polymerization or conjugation can be achieved by modifying the glycosaminoglycan with a heterobifunctional cross-linking reagent and linking the modified glycosaminoglycan to itself or to a desired core protein. Polymerized molecules may be useful due to their larger size. Intact heparan sulfate, i.e., repeating glucosamine and hexuronic acid units linked to a core protein in the ECM, may be particularly useful.

Suitable polypeptides that can affect (e.g., inhibit) TLR4 activity can include anti-CD14 polypeptides and antibodies (e.g., IC14, WT14, or ab8103). See, for example, U.S. Pat. No. 5,869,055, WO 02/42333, and WO 01/72993. Alternatively, analogues of agonists such as lipid-A, fibronectin EDA, fibrinogen, or taxol also can be used to inhibit TLR. For example, the lipid-A analogues alpha-D-glucopyranose, 3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-beta-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate) tetrasodium salt (E5564) and 6-O-[2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-beta-O-phosphono-alpha-D-glucopyranose tetrasodium salt (E5531) can be used to inhibit TLR4. See, Mullarkey et al. (2003) J. Pharmacol. Exp. Ther. 304(3):1093-102. In some embodiments, the test compound can be an antibiotic (e.g., geladamycin). See, Vega and Maio (2003) Mol. Biol. Cell 14:764-773.

Agents that alter TLR4, MD-2, or CD14 activity can be administered to a non-human animal by any route, including, without limitation, oral or parenteral routes of administration such as intravenous, intramuscular, intraperitoneal, subcutaneous, intrathecal, intraarterial, nasal, or pulmonary administration. A test compound can be formulated as, for example, a solution, suspension, or emulsion with pharmaceutically acceptable carriers or excipients suitable for the particular route of administration, including sterile aqueous or non-aqueous carriers. Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions. Examples of non-aqueous carriers include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Preservatives, flavorings, sugars, and other additives such as antimicrobials, antioxidants, chelating agents, inert gases, and the like also may be present.

For oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods known in the art. Preparations for oral administration can also be formulated to give controlled release of the compound. Nasal preparations can be presented in a liquid form or as a dry product. Nebulised aqueous suspensions or solutions can include carriers or excipients to adjust pH and/or tonicity.

Agents that alter TLR4, MD-2, and/or CD14 activity can be used to reduce body fat, increase percent lean body mass, increase bone density, and/or reduce bone loss in an animal. In general, one or more agents that alter TLR4, MD-2, or CD14 activity can be formulated as described above and administered to an animal in an amount effective to reduce body fat, increase percent lean body mass, increase bone density, and/or reduce bone loss. For example, compounds that inhibit TLR4 activity can be administered to farm animals such as pigs, turkeys, cows, chickens, goats, or sheep, or household pets such as cats or dogs to increase percent lean body mass. In general, leaner animals live longer and, in addition, leaner farm animals are useful in meat production. Subjects being treated with TLR inhibitors may have an increased susceptibility to infections. Thus, in some embodiments, antibiotics can be administered prophylactically to animals receiving TLR inhibitors to prevent the development of infections.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Body Mass of Mice Lacking TLR4

Lean body mass, body mass, percent body fat, and fat body mass in female mice lacking functional TLR4 (C3H/HeJ) were compared to the same characteristics of age and sex matched control mice having functional TLR4 (C3H/HeSnJ). C3H/HeJ and C3H/HeSnJ mice are commercially available from Jackson Labs. The results are presented in Table 1. Mice lacking functional TLR4 (C3H/HeJ) rarely gained more than 17% fat body mass, with the body fat that was present exhibiting a normal distribution. Although the mice were housed in cages, the C3H/HeJ mice had athletic bodies. In contrast, the control mice gained significantly more fat body mass. Lean body mass was less affected by the mutation in TLR4 than fat body mass.

These findings were confirmed by comparing a separate strain of mice with a different TLR4 mutation to its wild-type control strain. Mice in the second strain, C57B1/10ScNCr, contain a naturally occurring TLR deletion (a recessive deletion of the entire gene) and were purchased from the National Cancer Institute. The C57B1/10ScNCr mice also were significantly leaner than wild type and controls C57B1/10SnJ (Jackson Labs) (see Table 2).

Observation of body mass in a third mouse strain confirmed that the difference in body fat is TLR4-dependent. Mice in which the TLR4 mutation of C3H/HeJ was crossed onto a Balb/c mouse background (strain C.C3H-TLR4-lpsd, available from Jackson Labs) also had significantly less body fat and similar lean body mass at 6 weeks of age (see Table 3).

TABLE 1 TLR4 Mutant (C3H/HeJ) Measurement Age (wks) N P value Average St. Dev % of WT Lean Mass  8  5 0.28  14.26 g 1.08 93.2% Lean Mass 10 8-9 0.0021 13.53 g 1.22 90.0% Lean Mass 12 5-6 0.0946 15.37 g 1.04 92.2% Lean Mass    12 -B* 10 3.49 × 10⁻⁵   13.49 g 1.85 78.8% Lean Mass 24 15 0.0004 16.62 g 1.52 87.8% Lean Mass 31 14 3.7 × 10⁻⁸  16.01 g 1.13 82.6% Body Mass  8  5 0.11  16.20 g 1.24 85.3% Body Mass 10 8-9 0.0043 15.32 g 1.42 86.4% Body Mass 12 5-6 0.0205 18.47 g 1.40 77.5% Body Mass   12 -B 10 1 × 10⁻⁶ 15.27 g 2.19 70.4% Body Mass 24 15 1 × 10⁻⁷ 19.10 g 1.83 71.0% Body Mass 31 14 1.23 × 10⁻¹²   19.51 g 1.94 62.3% Fat Mass  8  5 0.038   1.96 g 0.18 53.6% Fat Mass 10 8-9  0.00081  1.78 g 0.29 66.3% Fat Mass 12 5-6  0.00978  3.10 g 0.46 69.2% Fat Mass   12 -B 10 4 × 10⁻⁷  1.78 g 0.44 38.9% Fat Mass 24 15 5 × 10⁻⁹  2.62 g 0.54 32.9% Fat Mass 31 14 1.70 × 10⁻¹³    3.49 g 0.99 29.2% % Fat  8  5 0.011  11.92% 0.32 63.4% % Fat 10 8-9 0.0026 11.57% 1.14 76.7% % Fat 12 5-6 0.0180 16.75% 1.53 79.3% % Fat 12   10 -B 1 × 10⁻⁶ 11.53% 1.85 55.4% % Fat 24 15  2 × 10⁻¹² 13.30% 1.36 45.8% % Fat 31 14 1.11 × 10⁻¹⁵   17.28% 2.93 45.6% *-B = live in the Barrier facility (sterile)

Each mouse strain was routinely tested for numerous infections, since infection can lead to loss of muscle and total body weight. No infections were observed and the mice continued to grow throughout the analysis. This was confirmed by comparing age and sex matched mice in the mouse facility with identical mice in the super sanitary Barrier facility. By the age of 12 weeks, the mice in the barrier facility showed the same TLR4 dependent body fat differences, in fact more so than those in the regular animal facility. All mice appeared healthy and reproduced effectively, with similar numbers of offspring to wild-type control mice. Taken together, these data indicate that TLR4 is a master regulator of fat body mass, and that loss of TLR4 signaling may result in inhibition of fat gain or in loss of body fat.

TABLE 2 TLR4 Deleted (C57Bl/10ScNCr) Measurement Age (wks) N P value Average St. Dev % of WT Lean Mass 6 10 0.0887 14.5875 g 1.0723 105.32% Lean Mass 9 5-6 0.0216 14.6667 g 0.4633 94.63% Lean Mass 20  4 0.792 20.5250 g 2.0105 98.43% Body Mass 6 10 0.710 16.6125 g 1.1281 100.99% Body Mass 9 5-6 0.0277 16.6333 g 0.4803 94.72% Body Mass 20  4 0.0917 25.2250 g 2.6763 87.74% Fat Mass 6 10 0.000477  2.0375 g 0.1061 78.37% Fat Mass 9 5-6 0.477  1.9833 g 0.1472 95.35% Fat Mass 20  4 0.00714  4.7500 g 0.7594 60.32% % Fat 6 10 0.000195 12.2875%   0.8202 78.07% % Fat 9 5-6 0.927 11.9% 0.7305 100.48% % Fat 20  4 0.00221 18.7% 1.2754 68.50%

TABLE 3 TLR4 Mutant Congenic on Balb/c background (C.C3H-TLR4-lpsd) Measurement Age (wks) N P value Average St. Dev % of WT Lean Mass 6 10 0.089 13.79 g 0.8749 105.19% Body Mass 6 10 0.809 15.95 g 0.9880 100.69% Fat Mass 6 10 0.000399  2.17 g 0.2406 79.49% % Fat 6 10 8 × 10⁻⁶ 13.5% 1.1695 78.48%

Example 2 Bone Density of Mice Lacking TLR4

Bone density, bone area, and bone calcium were examined in the three strains of TLR4 mutant mice described above and compared to that of age and sex matched control mice having a functional TLR4. Bone density, bone calcium content and bone area were measured by dual x-ray absorptometry using a PIXIMUS small animal densitometer (LUNAR, Madison, Wis.). Mice were either euthanized or anesthetized by IP injection according to IUCAC approved procedures. All measurements were taken in live anesthetized mice, or euthanized mice. Data analysis was done with PIXIMUS software. All bone measurements excluded the skull, as recommended by LUNAR. Tibia and femur measurements were obtained by measuring bone parameters within a region of interest surrounding the right or left tibia or femur of each mouse. The same skeletal landmarks were used to select the region of interest in both controls and mutant mice. As indicated in Tables 4-6, mice with mutations in TLR4 have significantly increased bone mineral density, bone mineral content, and bone area, as measured by dual x-ray absorptometry. TLR4 mutations lead to higher bone mineral density and higher bone mineral content despite similar total body weights. Given the strong positive correlation in mammals of body fat and bone mineral density, it was unexpected that these mutant mice would have higher bone density and lower % body fat. Mutant mice also have bones of larger area. These differences were not present in all of mice.

TABLE 4 TLR4 Mutant (C3H/HeJ) Measurement Age (wks) N P value Average St. Dev % of WT Bone Density 8  5 0.21 0.0467 g/cm² 0.0015 97.07% Bone Density 10 8-9 0.58 0.0454 g/cm² 0.0023 98.77% Bone Density 24 15 7.67 × 10⁻⁴ 0.0596 g/cm² 0.0024 105.08% Bone Density 31 14 1.59 × 10⁻⁵ 0.0620 g/cm² 0.0022 106.68% Bone Calcium 8  5 0.71 0.3600 g 0.0260 101.35% Bone Calcium 10 8-9 0.63 0.3342 g 0.0307 102.01% Bone Calcium 24 15 1.71 × 10⁻⁸ 0.5254 g 0.0286 117.21% Bone Calcium 31 14   0.00015 0.5211 g 0.0336 113.20% Bone Area 8  5 0.24 7.7040 cm² 0.3913 128.01% Bone Area 10 8-9 0.21 7.3500 cm² 0.3347 103.14% Bone Area 24 15 2.62 × 10⁻⁷ 8.8226 cm² 0.3992 111.80% Bone Area 31 14  0.0124 8.4507 cm² 0.4214 106.65%

TABLE 5 TLR4 Deleted (C57Bl/10ScNCr) Measurement Age (wks) N P value Average St. Dev % of WT Bone Density 6 10  0.3432 0.0425 g/cm² 0.0012 98.72% Bone Density 9 5-6 0.0980 0.0441 g/cm² 0.0014 96.90% Bone Density 20 4 0.270 0.0583 g/cm² 0.0033 103.92% Bone Calcium 6 10  0.3729 0.2988 g 0.0162 102.17% Bone Calcium 9 5-6 0.216 0.3305 g 0.0198 94.97% Bone Calcium 20 4 0.0254 0.5415 g 0.0611 121.34% Bone Area 6 10  0.0733 6.9813 cm² 0.2832 102.92% Bone Area 9 5-6 0.37225 7.4983 cm² 0.3092 97.51% Bone Area 20 4 0.00609 9.2600 cm² 0.5062 116.30% Femur Density 9 5-6 0.100 0.0593 g/cm² 0.0040 95.13% Femur Density 20 4 0.0351 0.1039 g/cm² 0.0108 111.38% Femur Calcium 9 5-6 0.0306 0.0192 g 0.0010 94.36% Femur Calcium 20 4 0.00845 0.0345 g 0.0046 120.00% Femur Area 9 5-6 0.846 0.3283 cm² 0.0175 100.41% Femur Area 20 4 0.0646 0.3325 cm² 0.0282 108.13% Tibia Density 9 5-6 0.00723 0.0477 g/cm² 0.0018 95.31% Tibia Density 20 4 0.00656 0.0719 g/cm² 0.0059 112.39% Tibia Calcium 9 5-6 0.0258 0.0195 g 0.0011 94.20% Tibia Calcium 20 4 0.00118 0.0321 g 0.0034 119.53% Tibia Area 9 5-6 0.743 0.4042 cm² 0.0202 99.30% Tibia Area 20 4 0.0931 0.4450 cm² 0.0169 105.01%

TABLE 6 TLR4 Mutant Congenic on Balb/c background (C.C3H-TLR4-lpsd) Measurement Age (wks) N P value Average St. Dev % of WT Bone Density 6 10 0.761 0.045 g/cm² 0.0022 99.28% Bone Calcium 6 10 0.165 0.2723 g 0.0304 106.70% Bone Area 6 10 0.0113 6.544 cm² 0.4337 107.35%

Example 3 CD14 Acts with TLR4 in Regulating Body Fat and Bone Density

To confirm the results showing decreases in body fat and increases in bone density and mineral content with a loss-of-function of TLR4, CD14 knockout mice (B6.129S-Cd14^(tmlFrm)) were analyzed and compared with the control strain, C57B1/6J, using dual x-ray absorptometry. CD14 knockout mice and C57B1/6J control mice were purchased from Jackson Labs. The CD14 knockout mice have been backcrossed 20 times onto the C57B1/6J strain. The TLR4 mutant phenotype of high bone mineral density and low percent body fat also was present in CD14 knockout mice (see Table 7). This indicates that the TLR/CD14 receptor complex regulates body fat and bone density. The fat mass and percent fat differences were significant at 6 weeks of age but were not significant at 12 weeks of age.

TABLE 7 CD14 Knock-out (B6.129S-Cd14^(tm1Frm)) Measurement Age (wks) N P value Average Value St. Dev % of WT Lean Mass 6 4-5 0.00957 12.98 g 0.46 106.3% Lean Mass 12 10 0.0000152 15.49 g 0.66 112.9% Body Mass 6 4-5 0.724 14.98 g 0.65 100.8% Body Mass 12 10 0.000231 18.46 g 0.90 110.8% Fat Mass 6 4-5 0.00180  1.96 g 0.18 73.5% Fat Mass 12 10 0.785  2.99 g 0.31 101.7% % Fat 6 4-5 0.000386 13.14% 1.01 73.5% % Fat 12 10 0.109 16.16% 1.09 91.9%

Bone density, bone calcium content, bone area, moment of inertia and moment of resistance of the mid-shaft (mid-diaphysis) of the right tibia (9.2 mm from the proximal end of each tibia) of the CD14 knockout mice were measured by peripheral quantitative computed tomography (pQCT) using a XCT Research SA+ pQCT scanner (STRATEC Medizinetechnik GmbH, Durlacher, Germany). All mice were 13 weeks and 5 days old, and were female. Mice were anesthetized by IP injection. Data analysis was done with STRATEC software version 5.40. The same skeletal landmarks were used in all measurements. Results are presented in Tables 8 and 9.

TABLE 8 CD14 Knock-out (B6.129S-Cd14^(tm1Frm)) Measurement Age (wks) N P value Average Value St. Dev % of WT Total Bone Density 6 4-5 0.0062  0.0424 g/cm² 0.0015 111.68% Total Bone Density 12 10 3.65 × 10⁻⁸ 0.0488 g/cm² 0.0013 110.44% Total Bone Calcium 6 4-5 0.0010  0.2820 g 0.0124 126.46% Total Bone Calcium 12 10 3.35 × 10⁻⁷ 0.3473 g 0.0013 121.73% Total Bone Area 6 4-5 0.00083 6.6520 cm² 0.1588 113.32% Total Bone Area 12 10 0.00019 7.1170 cm² 0.2915 110.20%

TABLE 9 CD14 Knock-out (B6.129S-Cd14^(tm1Frm)) Average Parameter Value St. Dev. N % of WT P Value Total Bone Content 0.916 mg 0.0368 9/9 109.87 0.00036 Cortical and Subcortical Bone 0.857 mg 0.0346 9/9 108.44 0.0015 Content Trabecular Bone Content 0.056 mg 0.0073 9/9 125.00 0.0019 Cortical Bone Content 0.690 mg 0.0300 9/9 109.52 0.0031 Total Bone Density 701.867 16.9031 9/9 97.21 0.031 mg/mm² Cortical and Subcortical Bone 853.556 13.7198 9/9 100.50 0.69 Density mg/mm² Trabecular Bone Density 190.600 8.5481 9/9 97.87 0.55 mg/mm² Cortical Bone Density 1091.800 15.8536 9/9 98.89 0.17 mg/mm² Total Bone Area 1.304 mm² 0.0702 9/9 112.78 0.00012 Cortical and Subcortical Bone 1.007 mm² 0.0381 9/9 108.11 0.0014 Area Trabecular Bone Area 0.299 mm² 0.0344 9/9 131.22 4.4 × 10⁵ Cortical Bone Area 0.631 mm² 0.0247 9/9 110.51 0.0012 Mean Cortical Thickness 0.182 mm  0.0040 9/9 103.41 0.14 Periosteal Circumference 4.048 mm  0.1079 9/9 106.30 9.0 × 10⁵ Endosteal Circumference 2.091 mm  0.1063 9/9 107.46 0.00057 Polar Moment of Inertia of Total 0.284 mm⁴ 0.0288 9/9 126.73 8.1 × 10⁵ Bone Polar Moment of Inertia of 0.143 mm⁴ 0.0150 9/9 132.99 4.4 × 10⁵ Cortical Bone Polar Moment of Inertia of 0.129 mm⁴ 0.0154 9/9 130.34 0.00026 Weighted Cortical Bone X* Axial Moment of Inertia of 0.143 mm⁴ 0.0158 9/9 132.99 3.0 × 10⁵ Total Bone X Axial Moment of Inertia of 0.071 mm⁴ 0.0105 9/9 142.22 0.00013 Cortical Bone X Axial Moment of Inertia of 0.067 mm⁴ 0.0087 9/9 139.53 9.0 × 10⁵ Weighted Cortical Bone Y Axial Moment of Inertia of 0.142 mm⁴ 0.0274 9/9 119.63 0.0039 Total Bone Y Axial Moment of Inertia of 0.071 mm⁴ 0.0071 9/9 123.08 0.0030 Cortical Bone Y Axial Moment of Inertia of  0.63 mm⁴ 0.0088 9/9 123.91 0.0031 Weighted Cortical Bone Polar Moment of Resistance of  0.33 mm³ 0.032 9/9 123.05 0.00041 Total Bone Polar Moment of Resistance of  0.22 mm³ 0.016 9/9 122.01 8.7 × 10⁵ Cortical Bone Polar Moment of Resistance of  0.19 mm³ 0.014 9/9 121.83 8.1 × 10⁵ Weighted Cortical Bone X Axial Moment of Resistance of  0.21 mm³ 0.027 9/9 123.33 0.0012 Total Bone X Axial Moment of Resistance of  0.12 mm³ 0.007 9/9 122.09 4.0 × 10⁵ Cortical Bone X Axial Moment of Resistance of  0.11 mm³ 0.009 9/9 123.38 0.00019 Weighted Cortical Bone Y Axial Moment of Resistance of  0.20 mm³ 0.020 9/9 116.99 0.0050 Total Bone Y Axial Moment of Resistance of  0.11 mm³ 0.008 9/9 116.09 0.0088 Cortical Bone Y Axial Moment of Resistance of  0.10 mm³ 0.007 9/9 116.88 0.0050 Weighted Cortical Bone *X axis is Anterior-Posterior, Y Axis is Lateral

Polar moment of resistance (by pQCT) and density (by dual X ray absorptometry) were well correlated with bone failure strength. Both of these parameters predicted significantly stronger bones in CD14 knockout animals. Taken as a whole, the dual X ray absorptometry and pQCT data indicate that the CD14 knockout mice have stronger bones then wild-type, but differ in their measurements of bone density. Bone density measurements by pQCT showed no difference, while bone density measurements by dual X ray absorptometry showed significant differences. Both dual X ray absorptometry and pQCT showed significantly more total bone content in CD14 knockout mice compared to wild-type controls.

Example 4 TLR4/CD14 Regulates Bone Stiffness and Resistance to Fracture

To confirm that the increased bone density and mineral content in the mutant mice correlates with actual increased bone strength, tibias from CD14 knockout mice were compared to control mice. Stiffness, elastic modulus and maximum force sustainable before fracture of tibias were measured by three-point biomechanical testing as follows. Mouse tibias were freshly dissected and mechanically tested in a 3-point bending configuration to determine their flexural properties. Testing was performed using a Dynamic Mechanical Analyzer (DMA 2980, New Castle, Del.). An increasing load was applied, at a rate of 0.1 N per second, to the anterior aspect of each tibia diaphysis until failure. Specimens were immersed in saline before and during testing. Using the Euler-Bernoulli beam formulation (eqn. 1), the slope of the force-deflection curve was used to calculate the bone's bending rigidity (EI).

where P=applied load, δ=beam deflection at mid-span, l=beam distance between outer supports, E=Young's modulus, I=area moment of inertia.

To determine material properties, each tibia was imaged by cross-section by pQCT using a XCT Research SA+ pQCT scanner (STRATEC Medizinetechnik GmbH, Durlacher, Germany). This cross-sectional data was used to calculate the moment of inertia (I) near the tibia mid-span using STRATEC software version 5.40. The moment of inertia was used in Equation 1 to determine the Young's modulus (E) in bending.

Tibias from mutant mice had increased stiffness and were able to bear a higher maximum load before fracture (see Table 10). This suggests that blockade of TLR4/CD 14 can result in changes in bone that reduce the incidence of fracture, as commonly occur in osteoporosis and other bone disorders. The elastic modulus of mutant bones was decreased, but this difference was not significant according to these tests. These data suggest that bones from mice with mutations in the TLR4/CD14 receptor complex have normal molecular architecture of their bones. This is as opposed to what is seen in osteopetrosis, where bones are denser, but are also more brittle. These data suggest that drug therapy targeted at inhibiting TLR4/CD14 for extended periods of time will result in increased bone density and strength without resulting in poor bone architecture, or brittleness.

TABLE 10 CD14 Knockout (B6.129S-Cd14^(tm1Frm)) Measurement Age (wks) N P value Average St. Dev % of WT Stiffness 4 8 0.00652 53.1 N/mm 8.17 114.15% Elastic Modulus 14 18 0.126 11.4 GPa 1.68 92.20% Maximum Force 4 8 6.33 × 10⁻⁷ 11.22 N 0.863 117.21%

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

1. A method for animal breeding, comprising: a) identifying a first animal as having a variant TLR4 nucleic acid or polypeptide; b) identifying a second animal as having a variant TLR4 nucleic acid or polypeptide; and c) breeding the first animal with the second animal to generate progeny.
 2. The method of claim 1, wherein the second animal is identified as having the same variant TLR4 nucleic acid or polypeptide as the first animal.
 3. The method of claim 1, wherein the second animal is identified as having a variant TLR4 nucleic acid or polypeptide that differs from the variant TLR4 nucleic acid or polypeptide of the first animal.
 4. The method of claim 1, wherein the animal is bovine, ovine, porcine, or fowl.
 5. The method of claim 1, further comprising breeding the progeny generated in step (c) to obtain animals that are homozygous for the variant TLR4 nucleic acid or polypeptide.
 6. The method of claim 5, further comprising measuring the level of body fat in the homozygous animals.
 7. A method for animal breeding, comprising: a) identifying a first animal as having a variant TLR4 nucleic acid or polypeptide; b) identifying a second animal as having a variant TLR4 nucleic acid or polypeptide; c) providing biological samples from the first animal and the second animal;| d) measuring the level of TLR4 activity in the biological samples; e) comparing the level of TLR4 activity in the biological samples from the first and second animals to a control level of TLR4 activity; and f) if the level of TLR4 activity is determined to be reduced in both the first animal and the second animal as compared to the control level of TLR4 activity, breeding the first animal with the second animal to generate progeny.
 8. The method of claim 7, wherein the first and second animals are treated with a TLR4 agonist prior to providing the biological samples.
 9. The method of claim 7, wherein the control level of TLR4 activity is the level of TLR4 activity in a corresponding control animal.
 10. The method of claim 7, wherein the second animal is identified as having the same variant TLR4 nucleic acid or polypeptide as the first animal.
 11. The method of claim 7, wherein the second animal is identified as having a variant TLR4 nucleic acid or polypeptide that differs from the variant TLR4 nucleic acid or polypeptide of the first animal.
 12. The method of claim 7, wherein the animal is bovine, ovine, porcine, or fowl.
 13. The method of claim 7, further comprising breeding the progeny generated in step (f) to obtain animals that are homozygous for the variant TLR4 nucleic acid or polypeptide.
 14. The method of claim 13, further comprising measuring the level of body fat in the homozygous animals.
 15. A method for animal breeding, comprising: a) identifying a first animal as having a reduced level of TLR4 activity as compared to a corresponding control animal; b) identifying a second animal as having a reduced level of TLR4 activity as compared to the corresponding control animal; and c) breeding the first animal with the second animal to generate progeny.
 16. The method of claim 15, wherein said animal is bovine, ovine, porcine, or fowl.
 17. The method of claim 15, wherein the level of TLR4 activity in the first or second animal is determined by measuring the expression of a nucleic acid.
 18. The method of claim 17, wherein the nucleic acid encodes a cytokine or a chemokine
 19. The method of claim 18, wherein the cytokine is an interleukin or TNF-α.
 20. The method of claim 18, wherein the chemokine is IP10.
 21. The method of claim 15, wherein said level of TLR4 activity in the first or second animal is determined by measuring the level of a TLR4 polypeptide.
 22. The method of claim 21, wherein the TLR4 polypeptide is measured in serum or in tissue.
 23. The method of claim 15, wherein the identifying steps comprise providing biological samples from the first animal and the second animal, measuring the level of TLR4 activity in the biological samples, and comparing the level of TLR4 activity in the first and second animals to a control level of TLR4 activity.
 24. The method of claim 23, wherein the first and second animals are treated with a TLR4 agonist prior to providing the biological samples.
 25. The method of claim 23, wherein the control level of TLR4 activity is the level of TLR4 activity in a corresponding control animal. 